CN109001337B - Method for detecting polysaccharide content of mesona blume - Google Patents

Abstract

The invention relates to a method for detecting the content of mesona blume polysaccharide, which comprises the following steps: preparing a series of mixed standard sample injection solutions with the concentration of 10-166.67 mu g/mL; preparing sample injection liquid of a sample to be detected; respectively detecting the prepared series of concentration mixed standard sample injection liquid and sample injection liquid of a sample to be detected by using a high performance liquid chromatography and evaporative light scattering detector combined method to obtain liquid chromatogram maps of the series of concentration mixed standard sample injection liquid and sample injection liquid of the sample to be detected; comparing the liquid phase map of the sample injection liquid of the sample to be detected with the liquid phase map of the sample injection liquid of each concentration standard sample, wherein the characteristic peak of the corresponding component with the same retention time as that in the liquid phase map of the sample injection liquid of the standard sample is the characteristic peak of the substance with the same component; and calculating the content of different components of polysaccharide in the mesona blume by a following standard curve according to the characteristic peak area.

Description

Method for detecting polysaccharide content of mesona blume

Technical Field

The present invention relates to testing or analysing materials by means of determining chemical or physical properties of the materials, and in particular to a method of detecting chemical components of plant polysaccharides.

Background

The Chinese mesona herb is an important plant resource used as both medicine and food, and the whole herb contains polysaccharide and has the functions of relieving summer heat, clearing heat, cooling blood and detoxifying. Mesona chinensis benth gum is a gel polysaccharide extracted from mesona chinensis benth and is often used in summer-heat-relieving products such as mesona chinensis benth, herbal tea and tortoise jelly, but when the viscosity is too high, the polysaccharide as a functional product can block diffusion and absorption in vivo, and the biological efficacy is difficult to fully exert. Because the polysaccharide has various types and existing forms and contents, for example, fungal polysaccharide has various biological activities such as antivirus, anticoagulation, blood fat reduction, antitumor, immunoregulation, aging delaying and the like, and has been developed into various medicinal and functional food additives, carrageenan is often used as a gelling agent, a thickening agent and an emulsifying agent to be added into food, and the like, the polysaccharide content in plants must be strictly and accurately detected and monitored in order to put mesona chinensis benth containing polysaccharide components into industrial production and achieve the standards of food and medicinal content.

At present, methods for detecting polysaccharide content mainly comprise a chemical method, a chromatography method, a capillary electrophoresis method, an infrared spectrum quantitative analysis method and the like, and the general development trend is that the detection process is simple, convenient, rapid and accurate, and trace detection and online detection are realized. The existing research shows that the chemical method for detecting the polysaccharide content has the advantages of simple operation and low cost, but the obtained total polysaccharide content cannot obtain the specific monosaccharide content; the components and the content of the polysaccharide can be qualitatively and quantitatively analyzed by chromatography, wherein a high performance liquid chromatography analysis method is a conventional analysis method, and can be used for performing constant and trace analysis on monosaccharide and oligosaccharide, but because the saccharide does not have ultraviolet absorption and fluorescence absorption, derivatization treatment needs to be performed on the saccharide, the high sensitivity requirement is required on a detector, the operation is relatively complex, and the cost is higher; the capillary electrophoresis method has the advantages of relatively simple instrument and less sample consumption, but in order to improve the separation efficiency, a surfactant is often used, and the operation process is relatively complicated; the infrared spectrum quantitative analysis method can realize rapid analysis, but the content of the polysaccharide component is calculated by measuring the intensity of the characteristic absorption band, and the measurement result is relatively inaccurate. The composition and the content of polysaccharide components in the mesona blume are respectively detected by adopting a high performance liquid chromatography, so that the content of each monosaccharide component in the mesona blume can be accurately detected, but if each monosaccharide component is respectively detected by the high performance liquid chromatography, the workload is very large. Therefore, a simple, accurate, efficient, low-cost and good-repeatability method for simultaneously detecting polysaccharide components and content in the mesona blume is urgently needed to be found for quality evaluation of the mesona blume medicinal material, and further promoting industrial production of the mesona blume medicinal material. At present, a detection method capable of simultaneously detecting the content of 10 polysaccharide components in the mesona blume does not exist.

Disclosure of Invention

The invention mainly solves the technical problem of providing the method for detecting the polysaccharide content of the mesona blume, can simultaneously and quantitatively detect 10 polysaccharides in the mesona blume, and has the advantages of simplicity, accuracy and high efficiency.

The technical scheme for solving the technical problems is as follows:

a method for detecting the content of mesona blume polysaccharide comprises the following steps:

(1) taking fructose, a-D-glucose, beta-D-glucose, D-ribose, sucrose, 2-methyl-alpha-D-fructofuranose, 2-methyl-beta-D-fructofuranose, beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, 1-ethyl-beta-D-glucose and 2-methyl-beta-D-fructopyranose respectively, adding methanol for dissolving, and then adding water for diluting to obtain a series of standard sample injection solutions with the concentration of 10-166.67 mu g/mL;

taking the mesona blume dry powder, adding 20 times of distilled water for ultrasonic extraction for 15min, filtering by a microporous membrane, volatilizing the solvent, and concentrating until each milliliter of concentrated solution contains 0.1 g of the mesona blume dry powder equivalent to each milliliter of concentrated solution, so as to obtain a sample solution of a sample to be detected;

and (3) respectively carrying out high performance liquid chromatography detection on the sample injection liquid of the sample to be detected and the sample injection liquid of each standard sample according to the following methods:

the sample amount is 10 muL, and the standard is 4.6mm multiplied by 250mm, 5μm,

the method comprises the steps of (1) introducing a sample into a chromatographic column with hydrophilic liquid chromatography as a filler, controlling the temperature of the column to be 25 ℃, then carrying out gradient elution by using a mobile phase consisting of 15-45% of phase A and 55-85% of phase B in volume ratio at the flow rate of 1.0mL/min, simultaneously detecting an eluent by adopting evaporative light scattering, controlling the temperature of a drift tube of an evaporative light scattering detector to be 60 ℃ and the flow rate of nitrogen to be 1.8L/min, and obtaining a high performance liquid chromatogram of mesona blume to be detected and high performance liquid chromatograms of a series of standard products; wherein, the A phase of the mobile phase is water; the phase B of the mobile phase is acetonitrile;

(2) comparing the liquid phase atlas of the sample injection liquid of the sample to be detected with the high performance liquid phase atlas of the sample injection liquid of the mixed standard sample with each concentration, wherein the characteristic peak of the corresponding component with the same retention time in the high performance liquid phase atlas of the sample injection liquid of the mixed standard sample is the characteristic peak of the substance with the same component; the contents of fructose, a-D-glucose, β -D-glucose, D-ribose, sucrose, 2-methyl- α -D-fructofuranose, 2-methyl- β -D-fructofuranose, β -D-galactose (1 → 6) - α -L-glucose (1 → 2) - β -D-fructose, 1-ethyl- β -D-glucose and 2-methyl- β -D-fructopyranose in mesona blume were calculated from the characteristic peak areas in a walking standard curve.

The detection method selects the standard sample injection liquid on the basis of separating and identifying 10 saccharide components in the mesona blume and selects the optimal detection parameters to ensure that the detected data of the polysaccharide content in the mesona blume is accurate and reliable. The detection method has the advantages of simplicity, rapidness, accuracy, high sensitivity, good precision, good stability and the like, and can simultaneously determine the content of fructose, a-D-glucose, beta-D-glucose, D-ribose, sucrose, 2-methyl-alpha-D-fructofuranose, 2-methyl-beta-D-fructofuranose, beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, 1-ethyl-beta-D-glucose and 2-methyl-beta-D-fructopyranose in the Chinese mesona herb sample. The method can be used for evaluating the quality of the mesona blume medicinal material, promotes the industrial development of the mesona blume medicinal material, and has the advantages of simplicity, accuracy, high efficiency, low cost and high sensitivity compared with the prior art.

Drawings

FIG. 1 is a high performance liquid chromatogram of Mesona chinensis Benth in an embodiment of the present invention, wherein A represents a liquid chromatogram of a sample to be tested; b represents a liquid phase map of the mixed standard sample injection liquid; in the A picture and the B picture, the peak 1 is fructose, the peak 2 is a-D-glucose, the peak 3 is beta-D-glucose, the peak 4 is D-ribose, the peak 5 is sucrose, the peak 6 is 2-methyl-alpha-D-fructofuranose, the peak 7 is 2-methyl-beta-D-fructofuranose, the peak 8 is beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, the peak 9 is 1-ethyl-beta-D-glucose, and the peak 10 is 2-methyl-beta-D-fructopyranose; the abscissa is retention time and the ordinate is peak height.

Detailed Description

Example 1

The content of 10 polysaccharide components in Chinese mesona herb is detected by using high performance liquid chromatography and an evaporative light scattering detector (HPLC-ELSD) together.

1. Instruments, reagents and samples

1.1. The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph (Agilent corporation, usa); alltech ELSD3300 evaporative light scattering detector (Alltech corporation, usa); sartorius CP225D analytical balance (d 0.01mg) (Sartorius, germany); millipore ultra pure water instruments (Millipore, USA).

1.2. Reagent: methanol (chromatographically pure, Honeywell, usa); acetonitrile (chromatographically pure, Honeywell, usa); formic acid (analytically pure, guangzhou chemical reagent house).

1.3. Sample (I)

A sample to be tested: selecting 16 batches of samples for detection, wherein the batches are A131201, A131204, A131205, A131210, A131213, A131214, A131215, A131217, A131218, A131219, A131220, A131221, A131222, A131223, A131224 and A131232 in sequence;

comparison products: fructose, a-D-glucose, beta-D-glucose, D-ribose, sucrose, 2-methyl-alpha-D-fructofuranose, 2-methyl-beta-D-fructofuranose, beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, 1-ethyl-beta-D-glucose and 2-methyl-beta-D-fructopyranose are all prepared by Guangzhou Chinese medicine university clinical pharmacology institute, and the purity is over 98 percent through area normalization method inspection. For the convenience of the following description, the above 10 saccharide components are sequentially described as: c-1, C-2, C-3, C-4, C-5, C-6, C-7, C-8, C-9 and C-10.

2. Method of producing a composite material

2.1. Preparing mixed standard sample injection liquid

Precisely weighing fructose, a-D-glucose, beta-D-glucose, D-ribose, sucrose, 2-methyl-alpha-D-fructofuranose, 2-methyl-beta-D-fructofuranose, beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, 1-ethyl-beta-D-glucose and 2-methyl-beta-D-fructopyranose respectively by 10mg, dissolving in distilled water by using a small amount respectively, fixing the volume to 2mL, and shaking up to obtain a standard sample injection solution.

Precisely sucking 10 mu L of the standard sample solutions, putting the sample solutions into a 10mL volumetric flask together, adding water to the volumetric flask until the volume is 0.30, 0.50, 0.80, 1.00, 1.25 and 5.00mL, and obtaining a series of mixed standard sample solutions.

2.2. Preparing sample injection liquid of to-be-detected product

Taking 1.0g of mesona blume medicinal material dry powder, sieving with a 80-mesh sieve, precisely weighing, placing in a triangular flask, adding 20mL of distilled water, carrying out ultrasonic extraction (200W, 40kHz) for 15min, shaking up, filtering with a 0.45-micron microporous membrane, collecting filtrate, volatilizing solvent, and fixing the volume to 10 mL.

2.3. High performance liquid chromatography combined with evaporative light scattering detector (HPLC-ELSD) for detection

Respectively injecting 10 mu L of mixed standard sample injection liquid and sample injection liquid of a sample to be detected into a high performance liquid chromatograph, and detecting eluent by evaporative light scattering to obtain a high performance liquid chromatogram.

The chromatographic conditions were as follows:

chromatograph: agilent 1260 high performance liquid chromatograph

A chromatographic column: MERCK ZIC-HILIC column (4.6mm x 250mm, 5 μm,

) (ii) a The chromatographic column uses hydrophilic liquid chromatography (Hilic) as filler

Mobile phase: the concentration by volume percent is water (A) -acetonitrile (B) gradient elution, and the gradient elution procedure is shown in Table 1.

TABLE 1 gradient elution of the mobile phase

Time (min)	0	10	20	40
					B％	85	85	70	55
A％	15	15	30	45

Flow rate: 1.0 mL/min;

column temperature: 25 ℃;

a detector: an Alltech ELSD3300 evaporative light scattering detector, the nitrogen flow rate is 1.8L/min, the drift tube temperature is 60 ℃, and the gain is 1;

the attribution of each peak in the mixed standard sample injection liquid chromatogram is determined by the following method: respectively preparing standard solutions of fructose, a-D-glucose, beta-D-glucose, D-ribose, sucrose, 2-methyl-alpha-D-fructofuranose, 2-methyl-beta-D-fructofuranose, beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, 1-ethyl-beta-D-glucose and 2-methyl-beta-D-fructopyranose, then respectively preparing high performance liquid chromatography maps of the single standard substance and the mixed standard substance sample injection liquid under the same chromatographic condition, and determining the attribution of each peak in the mixed standard sample injection liquid spectrum according to the retention time of the peak of the corresponding component in each single standard sample spectrum. After repeated experiments, the inventor finds that the components in the sample solution of the mixed standard substance in the mobile phase have fixed peak-appearance sequence, that is, from the left, the peak 1 is fructose, the peak 2 is a-D-glucose, the peak 3 is beta-D-glucose, the peak 4 is D-ribose, the peak 5 is sucrose, the peak 6 is 2-methyl-alpha-D-fructofuranose, the peak 7 is 2-methyl-beta-D-fructofuranose, the peak 8 is beta-D-galactose (1 → 6) -alpha-L-glucose (1 → 2) -beta-D-fructose, the peak 9 is 1-ethyl-beta-D-glucose, and the peak 10 is 2-methyl-beta-D-fructopyranose (see A picture in FIG. 1). Therefore, when the method is used, the attribution of each peak in the mixed standard sample injection liquid atlas can be determined according to the rule, the atlas of each standard sample injection liquid atlas does not need to be prepared, and time and reagents are saved.

2.4 Standard Curve and Linear relationship investigation

Taking the peak area measured by the polysaccharide component corresponding to each mixed standard sample injection liquid as the ordinate and the mass concentration as the abscissa, respectively making a series of scatter diagrams of 10 polysaccharide components in the standard sample injection liquid with concentration, and then taking the logarithm of the peak area of the obtained scatter diagrams (c) ((

) Performing two-variable linear regression on logarithm (X) of mass concentration to obtain regression equation

Wherein x is the logarithm of mass concentration (mg/ml), y is the logarithm of peak area (mAU), and a and b are constants; and confirming the peak-out position of the sample injection liquid of the sample to be detected according to the peak-out retention time of the sample injection liquid of the mixed standard sample, and substituting the peak area measured by the sample injection liquid of the sample to be detected into the correlation equation to calculate to obtain the content of 10 polysaccharide components in the sample solution.

The results showed that fructose, a-D-glucose, β -D-glucose, sucrose, β -D-galactose (1 → 6) - α -L-glucose (1 → 2) - β -D-fructose, and the peak area integrals thereof were in good linear relationship in the ranges of 0.14 to 4.48, 0.14 to 4.42, 0.24 to 7.54, 0.06 to 2.08, 0.07 to 2.12, 0.18 to 5.12, 0.16 to 6.34, 0.07 to 2.12, 0.21 to 6.64, and 0.17 to 4.83mg/ml, as shown in Table 2.

TABLE 2 Linear regression equation and Linear Range

2.5 minimum detection limit and minimum quantitation limit

The mixed standard sample solution was diluted with water and the detection was carried out according to the detection limit snr N of 3 and the detection limit snr N of 10, the results of which are shown in table 3.

TABLE 3 lowest detection limit and lowest quantitation limit

2.6 precision test

Precisely sucking the sample injection liquid of the same mixed standard substance, repeatedly injecting for 6 times, detecting, recording peak areas of C-1-C-10, and calculating RSD of the peak areas respectively to be 0.94%, 0.64%, 1.72%, 1.23%, 1.48%, 1.34%, 0.69%, 0.86%, 0.49% and 0.75%, which indicates that the precision of the instrument is good, and the result is shown in Table 4.

TABLE 4 results of precision test

2.7 stability test

Precisely weighing 1.0g of mesona blume medicinal material dry powder, sieving the powder by a 80-mesh sieve, preparing sample injection liquid of a sample to be tested according to a method marked under the item of '2.2', sequentially injecting samples for 0h, 2h, 4h, 8h, 16h and 24h respectively, measuring peak areas of C-1-C-10 and calculating the RSD of the content, wherein the peak areas are respectively 1.53%, 1.22%, 0.54%, 1.74%, 1.68%, 1.76%, 1.60%, 1.27%, 1.47% and 0.82%, which indicates that the sample to be tested is stable within 24h, and the result is shown in Table 5.

TABLE 5 stability test results

2.8 repeatability test

Precisely weighing 1.0g and 6 parts of a mesona blume medicinal material dry powder with the serial number of A131201, sieving by a 80-mesh sieve, preparing a sample injection liquid of a sample to be tested according to the method under item 2.2, precisely absorbing 10 mu L of the sample injection liquid for analysis, measuring the peak areas of C-1-C-10 and calculating the RSD of the content, wherein the RSD comprises the following components: 2.70%, 2.13%, 1.61%, 1.25%, 3.67%, 3.38%, 2.23%, 2.11%, 1.68%, 1.93%, indicating that the method has good reproducibility, and the results are shown in table 6.

TABLE 6 results of the repeatability tests

2.9 sample recovery test

Accurately weighing 0.5g and 6 parts of Mesona chinensis Benth medicinal material dry powder with the number of A131201, sieving with a 80-mesh sieve, sequentially adding appropriate amount of 10 reference substances, preparing sample injection solution of a sample to be tested according to the method under item 2.2, and performing sample injection analysis, wherein the result is shown in Table 7.

TABLE 7 sample recovery test results

2.10 measurement results of the content of 10 polysaccharide ingredients in Mesona chinensis Benth

Precisely weighing 1.0g of 16 batches of Chinese mesona herb dry powder, sieving the powder by a 80-mesh sieve, parallelly preparing 3 parts of sample injection liquid of a sample to be detected according to a method marked under the item '2.2', precisely sucking 10 mu L of the sample injection liquid for analysis, recording peak areas of C-1-C-10, and calculating the content, wherein the results are shown in a table 8.

Table 8 measurement results of 10 saccharide contents in mesona blume (n ═ 3)

Claims (1)

1. A method for detecting the content of mesona blume polysaccharide comprises the following steps:

(1) respectively taking fructose,a-D-glucose,β-D-glucose, D-ribose, sucrose, 2-methyl-alpha-D-fructofuranose, 2-methyl-beta-D-fructofuranose,β-D-galactose (1 → 6) -α-L-glucose (1 → 2) -βDissolving D-fructose, 1-ethyl-beta-D-glucose and 2-methyl-beta-D-fructopyranose in methanol, and diluting with water to obtain a series of standard sample injection solutions with the concentration of 10-166.67 mu g/mL;

taking the mesona blume dry powder, adding 20 times of distilled water for ultrasonic extraction for 15min, filtering by a microporous membrane, volatilizing the solvent, and concentrating until each milliliter of concentrated solution contains 0.1 g of the mesona blume dry powder equivalent to each milliliter of concentrated solution, so as to obtain a sample solution of a sample to be detected;

and (3) respectively carrying out high performance liquid chromatography detection on the sample injection liquid of the sample to be detected and the sample injection liquid of each standard sample according to the following methods:

according to the method, a chromatographic column with the specification of 4.6mm multiplied by 250mm, 5 μm and 200A and using hydrophilic liquid chromatography as a filler is subjected to sample injection according to the sample injection amount of 10 μ L, the temperature of the column is controlled to be 25 ℃, then a mobile phase consisting of 15-45% of an A phase and 55-85% of a B phase in volume ratio is used for carrying out gradient elution at the flow rate of 1.0mL/min, meanwhile, an evaporative light scattering detection eluent is adopted, the temperature of a drift tube of the evaporative light scattering detector is controlled to be 60 ℃, the nitrogen flow rate is 1.8L/min, and a high performance liquid chromatogram of mesona blume to be detected and high performance liquid chromatograms of a series of standard products are obtained; wherein, the A phase of the mobile phase is water; the phase B of the mobile phase is acetonitrile;

wherein the gradient elution procedure for the mobile phase is shown in the following table:

；

(2) comparing the liquid phase atlas of the sample injection liquid of the sample to be detected with the high performance liquid phase atlas of the sample injection liquid of the mixed standard sample with each concentration, wherein the characteristic peak of the corresponding component with the same retention time in the high performance liquid phase atlas of the sample injection liquid of the mixed standard sample is the characteristic peak of the substance with the same component; the contents of fructose, a-D-glucose, β -D-glucose, D-ribose, sucrose, 2-methyl- α -D-fructofuranose, 2-methyl- β -D-fructofuranose, β -D-galactose (1 → 6) - α -L-glucose (1 → 2) - β -D-fructose, 1-ethyl- β -D-glucose and 2-methyl- β -D-fructopyranose in mesona blume were calculated from the characteristic peak areas in a walking standard curve.

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